The study of Lyme disease has been hampered by the intractability of the etiological agent, Borrelia burgdorferi (Bb), to be genetically manipulated. This project will explore new avenues to improve the efficiency of genetically manipulating Bb; such developments will be applied towards understanding the role(s) of selected genes in virulence expression and/or colonization of hosts (ticks and mammals). The Specific Aims are: (1) To develop a novel insertion mutagenesis system (termed "RMEM") that will minimize the undesirable spontaneous loss of virulence that typically accompanies the genetic manipulation of Bb and (2) To apply the newly designed RMEM mutagenesis system to inactivate genes in a key regulatory network (e.g., rpoN-rpoS) and those involved in a stringent-like response (e.g. ndk, and spoT) to clarify their role(s) in regulating virulence expression and/or host colonization by Bb. This proposal thus seeks to contribute major new advances to borrelial genetics and to use those developments to examine potentially novel aspects of Bb virulence expression. [unreadable] [unreadable]